CN103592169B - A kind of method for identifying the wheat shore multiple alien substitution of wheat - Google Patents
A kind of method for identifying the wheat shore multiple alien substitution of wheat Download PDFInfo
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Abstract
A kind of initiative of the wheat shore multiple alien substitution of wheat comprising shore wheat 1Ns, 5Ns and 6Ns chromosome：By using (the 2n=8x=56 of Octoploid Tritileymus M842 12, AABBDDNsNs) with durum wheat (2n=4x=28, AABB) kind Trs 372 hybridizes, in F1, F2, F3 and F4 carries out cytology chromosome microscopy and genomic in situ hybridization (GISH) identification from generation to generation, screening chromosome number is 2n=42, and contain the individual plant of shore wheat Ns genome chromosomes, the equal bagging selfing of individual plant to filtering out, in F5 from generation to generation, 1 multiple alien substitution 05DM6 of wheat shore wheat containing shore wheat 1Ns, 5Ns and 6Ns chromosome is selected.
Description
Technical field
The present invention relates to a kind of initiative of wheat comprising shore wheat 1Ns, 5Ns and 6Ns chromosome-multiple alien substitution of shore wheat
And in situ hybridization, SSR and PAGE identifications.
Background technology
Wheat distance edge hybrid synthesizes new species using the abundant wheat genetic basis of exogenous genetic material and artificially creating
One of important channel and means.Shore wheat (Leymus mollis (Trin.) Hara), also known as soft bad grass, belong to grass family, barley
Subtribe, leymus ruderal, with drought resisting, resists cold, and Salt And Alkali Tolerance is barren-resistant, resists various fungies, and bacteriosis and stalk are thick
Strong, the characteristic such as big fringe is spent more, is the quality germplasm [2,3] of cereal cultivars improvement.The genome of shore wheat is
NsNsXmXm, wherein Ns genomes come from psathyrostachys huashanica (Psathyrostachys huashanica Keng), and Xm bases
Because the source organized not yet determines at present.
The sixties in last century, Tsitsin etc. obtain tetraploid, hexaploid wheat and Dalaicao, shore by IMMATURE EMBRYOS CULTURE
Wheat and the hybrid of Sha Shenglai grass.Anamthawat-J ó nsson etc. are doubled using embryo rescue and colchicin, are obtained common small
The hybrid amphidiploid of wheat, Triticum carthlicum and shore wheat, and 1 is reported comprising whole A and 1 B gene group chromosome, 1 pair of D gene
Group chromosome and the 6 couples of wheat of shore wheat chromosome-shore wheat amphidiploid materials A D99, are hybridized, sieve using AD99 and common wheat
Have selected 6 homozygous lines of mildew-resistance [2].Generation, gametes of the Li etc. (2005,2006) to Octoploid Tritileymus sporidiole
The formation of body and the genetic behavior of shore wheat chromosome are analyzed, and by using OCTOPLOID TRITICALE and Octoploid Tritileymus
Hybridization, obtains a wheat-rye comprising 6 chromosomess of rye and 6 shore wheat chromosomes-shore wheat Three genera.
Research by embryo save and colchicin chromosome doubling, obtain the big fringe with shore wheat, spend more, big grain, resist
The incomplete amphidiploid M842 of the common wheat of the characteristics such as disease-shore wheat；Hybridized using M842 and common wheat and Nullisomic wheat, training
3 alien addition lines and 2 alien substitutions are brought out；Using genomic in situ hybridization to the Octoploid Tritileymus of 6 types
The genome separation of (Octoploid Tritileymus) has carried out molecular cytogenetics analysis, by Octoploid Tritileymus
The genome separation of M842-4, M842-8, M842-12 and M842-16 is defined as AABBDDNsNs, the chromosome of M842-10
Group composition is defined as AABBDDNsNs+2Ta-2Lm (Ns), and the genome separation of M842-13 is defined as AABBDDJJ；Using eight
Times small shore wheat of body is hybridized with octoploid of wheat-wheatgrass, octoploid tuftlet wheat, obtain wheat-shore wheat-couchgrass, wheat-
Shore wheat-haynaldia villosa three bends hybrid [17,18].Using Octoploid Tritileymus M842-12 and M842-13 and durum wheat kind Trs-
372 and D4286 is hybridized, and CYTOGENETIC ANALYSIS OF ONE has been carried out to Hybrids F1.
In recent years, SSR molecular marker is in wheat genetic mapping, the assignment of genes gene mapping, pedigree or Genetic relationship, mark auxiliary
It is widely applied in the research such as exogenous genetic material identification in selection, wheat genetic background.Etc. establishing the first of wheat
Microsatellite genetic map is opened, they plan 230 the 279 of primer pair amplifies integrations to international wheat cluster mapping
(ITIM) on " Opata85 × W7984 " mapping population heredity frame diagram；Pestsova etc. is to the D bases that from aegilops tauschii separate
Because a group specialization microsatellite marker (GDM) is mapped on wheat, by " the Opata85 of 55 SSR integrations to ITIM
On × W7984 " mapping populations heredity frame diagram.International wheat microsatellites association (Wheat
MicrosatelliteConsortium, WMC) also will develop 66 WMC mark be incorporated into existing wheat genetic frame diagram
On；Existing nearly thousand SSR markers are positioned in wheat genetic physical map so far.The exogenic heredity thing in wheat genetic background
Matter identification research aspect, Guo etc. are using SSR marker to the racemosus in Triticum aestivum-Leymus multicaulis alien addition lines Line24
Rely grass chromosome to be identified, obtain 4 molecular labelings for identifying Leymus multicaulis chromosome in Line24, and according to
This 4 microsatellite markers are in the position of chromosome of wheat, thus it is speculated that the 1 pair of Leymus multicaulis chromosome added in Line24 is the
3rd, 5,6,7 homoeologous group chromosome reciprocal translocations are formed.SSR is to wheat-shape of tail goatweed for the application such as Peil
(Aegilopsmarkgrafii) exogenous chromosome in alien addition line is analyzed and identified, and obtain 22 can be common
The chromatinic molecular labeling of shape of tail sheep's hay is identified in Wheat Background；Iqbal etc. identifies wheat-mono- awns goatweed using SSR
(Aegilopsuniaristata) 3N chromosomes alien addition line.
The content of the invention
In the filial generation of Trs-372 × M84212,1 chromosome number has been filtered out for 2n=42, contained 6 shores
The strain 05DM6 of wheat chromosome, the present invention is using morphology, cytogenetics, genomic in situ hybridization, polyacrylamide gel
The technology such as electrophoresis and SSR molecular marker is studied the strain, to understand the morphological feature of strain 05DM6, genetic behavior,
Chromosome constitute, and contained shore wheat chromosome homologous group return it is in the wrong etc., be wheat breed improvement and chromosome engineering breeding provide
New germ plasm resource.
An object of the present invention is achieved through the following technical solutions：By using Octoploid Tritileymus M842-
12 (2n=8x=56, AABBDDNsNs) and durum wheat (2n=4x28, AABB) kind Trs-372 hybridizes, in F1, F2, F3
Cytology chromosome microscopy and genomic in situ hybridization (GISH) identification are carried out from generation to generation with F4, and screening chromosome number is 2n=
42, and containing the individual plant of shore wheat Ns genome chromosomes, the equal bagging selfing of individual plant to filtering out, selects 1 in F5 from generation to generation
The multiple alien substitution 05DM6 of wheat containing shore wheat 1Ns, 5Ns and 6Ns chromosome-shore wheat.
Another object of the present invention is a kind of method for identifying wheat-multiple alien substitution of shore wheat, it is characterised in that bag
Include following：
CYTOGENETIC ANALYSIS OF ONE：The F5 offsprings multiple alien substitution 05DM6 of small shore wheat of Trs-372 × M842-12, seed chamber
Interior germination, whne root it is long to 1~2cm when, the clip tip of a root is fixed in 0~4 DEG C of frozen water pretreatment 24h, Kano fixer, 1% fiber
The plain pectinase enzymatic hydrolysis of enzyme+1%, aceto-camine dyeing compressing tablet；Pollen mother cell is observed：Field takes of the right age young fringe, is fixed with Kano
Liquid is fixed, aceto-camine dyeing compressing tablet, the micro- sem observations of Olympus BX60；
Genomic in situ hybridization：DNA is extracted and probe mark：Shore wheat and psathyrostachys huashanica full genome are extracted using CTAB methods
Group DNA is simultaneously marked, chromosome sectioning and in situ hybridization, every film-making add 40 μ l hybridization solutions (comprising 20 × SSC4 μ l,
The 1 μ l of μ l, 10% (W/V) SDS (dodecyl sodium sulfate) of ssDNA (salmon sperm dna, 5ug/uL) 1,50% (W/V) DS (sulfuric acid Portugals
Glycan) 8 μ l, the μ l of deionized formamide 20, DNA probe 100ng, aseptic deionized water adds to 40 μ l).95 DEG C are denatured 8min, miscellaneous
Hand on hybrite in situ hybridization instrument according to program：75 DEG C of 8min, 37 DEG C of 16h are carried out；
SSR marker is analyzed：The μ l of pcr amplification reaction system 20, comprising 1 × buffer (100mM Tris-HCl pH8.3,
1.5mM MgCl2), 0.2M dNTPs, 50ng SSR primers, 1U Taq archaeal dna polymerases, 50~100ng template DNAs, 94 DEG C are pre-
Denaturation 3min, according to 94 DEG C of denaturation 1min, 50,55 or 60 DEG C of annealing 1min, 72 DEG C extend 2min and carry out, and finally fill for 34 circulations
Divide and extend 10min, amplified production carries out electrophoresis, μ l, the 150V constant-pressure conditions of point sample amount 8 through 8% non-denaturing polyacrylamide gel
4~5h of lower electrophoresis, cma staining, digital photo camera preserves result；
PAGE is analyzed：12% separation gel and 4.0% concentration glue is prepared, point sample amount is 10 μ l, electrode buffer
PH8.3, per plate glue constant current 15mA, 15 DEG C of electrophoresis 5h, coomassie brilliant blue R_250 stained over night, originally water decolorization, digital phase
Machine is taken a picture, gel strength 12%, gel cross-linkage degree 3.3%, point sample amount 10 μ l, electrode buffer pH3.1, per plate glue constant voltage
200V, is adjusted to 400V after electrophoresis 30min, electrophoresis 5h, 10% solution of trichloroacetic acid is fixed, coomassie brilliant blue R_250 dyeing, originally
Water decolorization, digital photo camera；
Morphological observation：Statistics Octoploid Tritileymus M842-12, durum wheat kind Trs-372, small shore wheat multiple different generation
The plant height and tiller number of 10 individual plants for being 05DM6 and Common Wheat Varieties 7182 are changed, the typical individual plant institute of each material 5 is investigated
There are stipes length, boot leaf size proterties under spike length, spikelet number, grain number per spike, the fringe of tiller, using SPSS V13.0 softwares to each property
The significance of difference of shape value carries out variance analysis.
Brief description of the drawings
Tip of a root somatic chromosome (a) of the multiple alien substitution 05DM6 of the small shore wheat of accompanying drawing 1 and its with pollen mother cell subtrahend
Metaphase I chromosomes (b)
The genomic in situ hybridization identification of the tip of a root somatic chromosome of the multiple alien substitution 05DM6 of the small shore wheat of accompanying drawing 2
(a) and the in situ hybridization of pollen mother cellses mid-term I DNA sequences identification (b)
The SSR molecular marker analysis of the multiple alien substitution 05DM6 of the small shore wheat of accompanying drawing 3
SDS-PAGE (a) and A-PAGE (b) identification of the multiple alien substitution 05DM6 of the small shore wheat of accompanying drawing 4 and its parent
The plant (a) of the multiple alien substitution 05DM6 of the small shore wheat of accompanying drawing 5 and its parent, tassel (b) and little Hua (c)
Specific embodiment
The preferred embodiments of the present invention will be below described in detail；It should be appreciated that preferred embodiment is only for saying
The bright present invention, rather than in order to limit the scope of the invention.
Saved by embryo and colchicin chromosome doubling, obtain the big fringe with shore wheat, spend more, it is big grain, disease-resistant etc.
The incomplete amphidiploid M842 of the common wheat of characteristic-shore wheat；Hybridized using M842 and common wheat and Nullisomic wheat, cultivated
3 alien addition lines and 2 alien substitutions；Using genomic in situ hybridization to the Octoploid Tritileymus of 6 types
The genome separation of (Octoploid Tritileymus) has carried out molecular cytogenetics analysis, by Octoploid Tritileymus
The genome separation of M842-4, M842-8, M842-12 and M842-16 is defined as AABBDDNsNs, the chromosome of M842-10
Group composition is defined as AABBDDNsNs+2Ta-2Lm (Ns), and the genome separation of M842-13 is defined as AABBDDJJ；Using eight
Times small shore wheat of body is hybridized with octoploid of wheat-wheatgrass, octoploid tuftlet wheat, obtain wheat-shore wheat-couchgrass, wheat-
Shore wheat-haynaldia villosa Three genera.By using Octoploid Tritileymus M842-12 (2n=8x=56, AABBDDNsNs) and solids
Wheat (2n=4x=28, AABB) kind Trs-372 hybridizes, and in F1, F2, F3 and F4 carry out cytology chromosome microscopy from generation to generation
With genomic in situ hybridization (GISH) identification, screening chromosome number is 2n=42, and contains shore wheat Ns genome chromosomes
Individual plant, the equal bagging selfing of individual plant to filtering out, in F5 from generation to generation, select 1 it is small containing 6 shore wheat Ns genome chromosomes
The multiple alien substitution 05DM6 of wheat-shore wheat.
Another object of the present invention is a kind of method for identifying wheat-multiple alien substitution of shore wheat, it is characterised in that bag
Include following：
CYTOGENETIC ANALYSIS OF ONE：The F5 offsprings multiple alien substitution 05DM6 of small shore wheat of Trs-372 × M842-12, seed chamber
Interior germination, whne root it is long to 1~2cm when, the clip tip of a root is fixed in 0~4 DEG C of frozen water pretreatment 24h, Kano fixer, 1% fiber
The plain pectinase enzymatic hydrolysis of enzyme+1%, aceto-camine dyeing compressing tablet；Pollen mother cell is observed：Field takes of the right age young fringe, is fixed with Kano
Liquid is fixed, aceto-camine dyeing compressing tablet, the micro- sem observations of OlympusBX60；Using M842-12 (AABBDDNsNs, 2n=56)
With durum wheat kind Trs-372 (AABB, 2n=28) hybridization, offspring through the bagging selfing of 4 generations, in F5 offsprings, by the tip of a root
Somatic chromosome microscopy, screens 1 plant of plant 05DM6 of 2n=42, harvests the individual plant, and indoor germination has been planted to its 15,
Crop field is seeded in after the fixed tip of a root；15 tip of a root chromosome number of somatic of seed are all 2n=42 (Fig. 1 a).Choose in field
54 pollen mother cellses mid-term I chromosome pairings situations are carried out microscopy observation, as a result by the of the right age young fringe of 05DM6
It has been shown that, 05DM6 has 46 chromosome configurations of pollen mother cell for 2n=21II, accounts for the 85.19% of observation of cell number；In divalence
In body, cyclic divalent body occupies very at high proportion, and average each cell has 18.97 (Fig. 1 b).
Genomic in situ hybridization：DNA is extracted and probe mark：Shore wheat and psathyrostachys huashanica full genome are extracted using CTAB methods
Group DNA is simultaneously marked, chromosome sectioning and in situ hybridization, every film-making add 40 μ l hybridization solutions (comprising 20 × SSC4 μ l,
The 1 μ l of μ l, 10% (W/V) SDS (dodecyl sodium sulfate) of ssDNA (salmon sperm dna, 5ug/uL) 1,50% (W/V) DS (sulfuric acid Portugals
Glycan) 8 μ l, the μ l of deionized formamide 20, DNA probe 100ng, aseptic deionized water adds to 40 μ l).95 DEG C are denatured 8min, miscellaneous
Hand on hybrite in situ hybridization instrument according to program：75 DEG C of 8min, 37 DEG C of 16h are carried out；
Genome is carried out by probe of shore wheat and psathyrostachys huashanica complete genome DNA respectively to the tip of a root body cell of 05DM6
FISH (GTSH) identifies, as a result shows that 05DM6 cells are inferred all containing 6 complete yellow green chromosome signals
05DM6 contains 6 Ns genome chromosomes (Fig. 2 a).It is female to its pollen thin using psathyrostachys huashanica total genomic dna as probe
Born of the same parents carry out genomic in situ hybridization identification, as a result show, 05DM6 pollen mother cellses mid-term I cells have 3 completely
Bivalent show yellowish green chrominance signal, show that 6 Ns genome chromosomes in 05DM6 can completely be paired into 3 pairs of chromosomes
(Fig. 2 b).Thus illustrate, 05DM6 is 1 inheritance stability, comprising 36 chromosomes of wheat and 3 pairs of shore wheat Ns genome dyeing
The wheat of the body-multiple alien substitution of shore wheat.
SSR marker is analyzed：To determine the genome of 05DM6 into using the wheat D genomes 1D of the announcements such as Pestsova
14 pairs of SSR primers of~7D chromosomes, PCR molecular marker analysis have been carried out to 05DM6 and its parent.Pcr amplification reaction system
20 μ l, comprising 1 × buffer (100mM Tris-HCl pH8.3,1.5mM MgCl2), 0.2M dNTPs, 50ng SSR primers,
1U Taq archaeal dna polymerases, 50~100ng template DNAs, 94 DEG C of predegeneration 3min, 34 circulations according to 94 DEG C of denaturation 1min, 50,
55 or 60 DEG C of annealing 1min, 72 DEG C extend 2min and carry out, and last fully to extend 10min, amplified production is through 8% non denatured polypropylene
Acrylamide gel carries out electrophoresis, and 4~5h of electrophoresis under μ l, the 150V constant-pressure conditions of point sample amount 8, cma staining, digital photo camera is protected
Deposit result.
SSR interpretations of result show, the 2D of D genomes, the primer on 3D, 4D and 7D chromosome, and in 05DM6, octoploid is small
All amplify the set goal band in shore wheat M842 and wheat breed 7182, and the primer on 1D, 5D and 6D chromosomes, though
So the set goal band has been amplified in Octoploid Tritileymus M842 and wheat breed 7182, but do not have in 05DM6
The set goal band is amplified, the 1D of the wheat D genomes that illustrated what 05DM6 may be lacked, 5D and 6D chromosomes, and contain
The 2D of wheat D genomes, 3D, 4D and 7D chromosome (Fig. 3).The 1D of wheat D genomes in 05DM6,5D and 6D chromosomes may
Replaced by 1Ns, 5Ns and 6Ns chromosome of shore wheat.
PAGE is analyzed：Seed storage protein to 05DM6 and its parent has carried out SDS-polyacrylamide
Gel electrophoresis (SDS-PAGE) and acidity-polyacrylamide gel electrophoresis (A-PAGE) are identified.Prepare 12% separation gel and
4.0% concentration glue, point sample amount is 10 μ l, electrode buffer pH8.3, per plate glue constant current 15mA, 15 DEG C of electrophoresis 5h, examines horse
This light blue R-250 stained over night, originally water decolorization, digital photo camera, gel strength 12%, gel cross-linkage degree 3.3%, point sample
10 μ l, electrode buffer pH3.1 are measured, per plate glue constant voltage 200V, 400V, electrophoresis 5h, 10% trichlorine is adjusted to after electrophoresis 30min
Acetic acid solution is fixed, coomassie brilliant blue R_250 dyeing, originally water decolorization, digital photo camera；SDS-PAGE electrophoresis results show
Show, in high-molecular-weight glutelin area, 05DM6 and its external source parent M842-12 and shore wheat have 1 special HMW paddy egg
Bai Yaji (HMW-GS) and low-molecular-weight glutenin subunit (LMW-GS) band, and in control China spring, the wheat parent of 05DM6
Trs-372 and 7182 no band, while 05DM6 has also lacked wheat parent 71821D Chromosome G lu-D1 sites institute table
High-molecular-weight glutelin subunit (HMW-GS) band (Fig. 4 a, shown in arrow) for reaching；A-PAGE electrophoresis results show, in ω -ol
Molten protein domain, 05DM6 and its external source parent M842-12 and shore wheat have 1 special alcohol soluble protein band, and 05DM6
Wheat parent Trs-372 and 7182 no bands (Fig. 4 b, shown in arrow).Illustrate, 05DM6 is carried from shore wheat Ns bases
Because of special HMW-GS, LMW-GS and alcohol soluble protein band for organizing.Show, the 1D dyeing private savings of wheat D genomes are by shore in 05DM6
The 1Ns chromosomes of wheat Ns genomes are replaced.
Morphological observation：Statistics Octoploid Tritileymus M842-12, durum wheat kind Trs-372, small shore wheat multiple different generation
The plant height and tiller number of 10 individual plants for being 05DM6 and Common Wheat Varieties 7182 are changed, the typical individual plant institute of each material 3 is investigated
There are stipes length, boot leaf size proterties under spike length, spikelet number, grain number per spike, the fringe of tiller, using SPSS V13.0 softwares to each property
The significance of difference of shape value carries out variance analysis.Result shows that 05DM6 has the morphological feature of parents, and plant height substantially becomes short,
All lower than parents, fringe shape is closer to parent M842-12, is all spindle (Fig. 5 b), and sterile lemma and parent's Trs-372 phases
Seemingly, more smooth and with more obvious keel projection, the length of awns substantially shortens, is closer to parent M842-12.
The tiller number of 05DM6, boot leaf size, spike length, spikelet number and setting percentage are between parents, and stipes is long under plant height, fringe
The proterties such as degree and grain number per spike is substantially less than parents.Variance analysis shows, 05DM6 and parent M842-12, Trs-372,7182 phases
Than, difference in plant height significantly, tiller number, spike length, spikelet number and the Traits change such as stipes length is extremely notable under fringe, boot leaf size and knot
Real rate difference is not notable.
Claims (1)
1. it is a kind of identify wheat-multiple alien substitution of shore wheat method, it is characterised in that including analysis below：
1) CYTOGENETIC ANALYSIS OF ONE：The F5 offsprings multiple alien substitution 05DM6 of small shore wheat of Trs-372 × M842-12, in seed chamber
Germination, whne root it is long to 1~2cm when, the clip tip of a root is fixed in 0~4 DEG C of frozen water pretreatment 24h, Kano fixer, 1% cellulose
The pectinase enzymatic hydrolysis of enzyme+1%, aceto-camine dyeing compressing tablet；Pollen mother cell is observed：Field takes of the right age young fringe, uses Kano fixer
It is fixed, aceto-camine dyeing compressing tablet, the micro- sem observations of Olympus BX60, using M842-12 (AABBDDNsNs, 2n=56) with
Durum wheat kind Trs-372 (AABB, 2n=28) hybridize, offspring through the bagging selfing of 4 generations, in F5 offsprings, by tip of a root body
Cell chromosome microscopy, screens 1 plant of plant 05DM6 of 2n=42, harvests the individual plant, to its 15 seed indoor germinations, Gu
Crop field is seeded in after determining the tip of a root；15 tip of a root chromosome number of somatic of seed are all 2n=42, and the suitable of 05DM6 is chosen in field
54 pollen mother cellses mid-term I chromosome pairings situations are carried out microscopy observation by age children's fringe, are as a result shown, 05DM6
There are 46 chromosome configurations of pollen mother cell for 2n=21II, account for the 85.19% of observation of cell number；In bivalent, ring-type
Bivalent occupies very at high proportion, and average each cell has 18.97；
2) genomic in situ hybridization：DNA is extracted and probe mark：Shore wheat and psathyrostachys huashanica full-length genome are extracted using CTAB methods
DNA is simultaneously marked, chromosome sectioning and in situ hybridization, and every film-making adds 40 μ l hybridization solutions, the 40 μ l hybridization solutions comprising 20 ×
SsDNA (salmon sperm dna) 1 μ l, 10% (W/V) SDS (dodecyl sodium sulfate) 1 the μ l, 50% (W/V) of SSC4 μ l, 5ug/uL
The μ l of DS (dextran sulfate) 8, the μ l of deionized formamide 20, DNA probe 100ng, aseptic deionized water adds to 40 μ l, 95 DEG C of changes
Property 8min, hybridization on hybrite in situ hybridization instrument according to program：75 DEG C of 8min, 37 DEG C of 16h are carried out, to the tip of a root body of 05DM6
Cell carries out genome breeding (GISH) identification by probe of shore wheat and psathyrostachys huashanica complete genome DNA respectively,
Result shows that 05DM6 cells infer that 05DM6 contains 6 Ns genomes dyes all containing 6 complete yellow green chromosome signals
Colour solid, using psathyrostachys huashanica total genomic dna as probe, genomic in situ hybridization identification is carried out to its pollen mother cell, knot
Fruit shows that 05DM6 pollen mother cellses mid-term I cells have 3 complete bivalents to show yellowish green chrominance signal, show
6 Ns genome chromosomes in 05DM6 can completely be paired into 3 pairs of chromosomes, and 05DM6 is 1 inheritance stability, comprising 36
Bar chromosome of wheat and the 3 pairs of wheat of shore wheat Ns genome chromosomes-multiple alien substitutions of shore wheat；
3) SSR marker analysis：The μ l of pcr amplification reaction system 20, comprising 1 × buffer, 0.2M dNTPs, 50ng SSR primers,
1U Taq archaeal dna polymerases, 50~100ng template DNAs, the buffer is 100mMTris-HCl pH8.3,1.5mM
MgCl2, 94 DEG C of predegeneration 3min, 34 circulations are according to 94 DEG C of denaturation 1min, 50,55 or 60 DEG C of annealing 1min, 72 DEG C of extensions
2min, last fully to extend 10min, amplified production carries out electrophoresis through 8% non-denaturing polyacrylamide gel, the μ l of point sample amount 8,
4~5h of electrophoresis under 150V constant-pressure conditions, cma staining, digital photo camera preserves result, and SSR interpretations of result show, D genes
The 2D of group, the primer on 3D, 4D and 7D chromosome, in 05DM6, is all expanded in Octoploid Tritileymus M842 and wheat breed 7182
Go out the set goal band, and the primer on 1D, 5D and 6D chromosomes, although in Octoploid Tritileymus M842 and wheat breed
The set goal band is amplified in 7182, but the set goal band has not been amplified in 05DM6；
4) PAGE analyses：Prepare 12% separation gel and 4.0% concentration glue, point sample amount be 10 μ l, electrode buffer pH8.3,
Per plate glue constant current 15mA, 15 DEG C of electrophoresis 5h, coomassie brilliant blue R_250 stained over night, originally water decolorization, digital camera shines
Phase, gel strength 12%, gel cross-linkage degree 3.3%, point sample amount 10 μ l, electrode buffer pH3.1, per plate glue constant voltage
200V, is adjusted to 400V after electrophoresis 30min, electrophoresis 5h, 10% solution of trichloroacetic acid is fixed, coomassie brilliant blue R_250 dyeing, originally
Water decolorization, digital photo camera, SDS-PAGE electrophoresis results show, in high-molecular-weight glutelin area, 05DM6 and its external source parent
M842-12 and shore wheat have 1 special high-molecular-weight glutelin subunit (HMW-GS) and low-molecular-weight glutenin subunit (LMW-
GS) band, and in control China spring, the wheat parent Trs-372 of 05DM6 and 7182 no bands, 05DM6 has lacked small
High-molecular-weight glutelin subunit (HMW-GS) band expressed by wheat parent 71821D Chromosome G lu-D1 sites；
5) morphological observation：Statistics Octoploid Tritileymus M842-12, durum wheat kind Trs-372, the small multiple different replacement of shore wheat
It is the plant height and tiller number of 10 individual plants of 05DM6 and Common Wheat Varieties 7182, investigates the typical individual plant of each material 5 and own
Stipes length, boot leaf size proterties under spike length, spikelet number, grain number per spike, the fringe of tiller, using SPSS V13.0 softwares to each proterties
The significance of difference of value carries out variance analysis, and 05DM6 has the morphological feature of parents, and plant height becomes short, fringe shape all lower than parents
It is close with parent M842-12, all it is spindle, and sterile lemma is smooth and with keel projection, the length of awns shortens, with parent
M842-12 is approached, and the tiller number of 05DM6, boot leaf size, spike length, spikelet number and setting percentage are between parents.
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| CN111397999B (en) * | 2020-05-09 | 2021-12-03 | 中国科学院植物研究所 | Simple method for enhancing contrast of micro-CT plant sample |
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| CN1336105A (en) * | 2000-08-05 | 2002-02-20 | 广西黑五类食品集团公司 | Breeding process of high-quality black wheat 76 |
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| ATE495671T1 (en) * | 2007-07-19 | 2011-02-15 | Univ Montana State | FUNGAL ISOLATES AND THEIR USE FOR PROMOTING SALT AND DRY TOLERANCE IN PLANTS |
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| CN1336105A (en) * | 2000-08-05 | 2002-02-20 | 广西黑五类食品集团公司 | Breeding process of high-quality black wheat 76 |
| CN101643734A (en) * | 2009-09-14 | 2010-02-10 | 西北农林科技大学 | IF2 translation initiation factor gene relevant to wheat yellow rust resistance |
| CN102090318A (en) * | 2009-12-11 | 2011-06-15 | 陈红余 | Research for hybrid multiple-category wheat |
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